Inoue et al in Eur. J. Biochem. 2004, 271, 356-366, described the secretion of native silk fibroin of Bombyx mori from the posterior silk gland as a 2.3 MDa elementary unit (hereinafter termed EU) which consists of six sets of a disulfide-linked heavy chain-light chain fibroin heterodimer and one molecule of P25.
Currently, solutions of silk proteins (such as fibroin and other proteins) are made as regenerated silk solutions out of cocoons or silk threads which are dissolved in solubilisation agents (e.g. lithium bromide) and refolded through dialysis or other buffer exchange techniques (see, for example, Altmann et al., Biomaterials 2003, 24, 401-416). Given the known challenges in producing active proteins through refolding after solubilisation in protein folding/chaotropic agents (Vallejo and Rinas, Microbial Cell Factories, 2004, doi:10.1186/1475-2859-3-11), a person skilled in the art will appreciate the technical hurdles which have to be solved in order to produce correctly folded, native fibroin EUs from regenerated silk solutions. It is therefore not surprising that there has been no report to date on the successful production of high molecular weight fibroin assembled in EU conformation out of regenerated silk solutions.
The differences between the native silk proteins as produced and stored in the glands of arthropods, such as silkworms, (i.e. in their high molecular weigh EU conformation) and the regenerated silk proteins (disclosed above) are such that the regenerated silk proteins produced by current techniques have at most “native-like” features, i.e. the regenerated silk proteins have some properties in common but cannot be said to be identical or substantially identical with the native silk proteins. The native silk proteins are defined as those proteins found in their native protein conformation, i.e. with the primary, secondary, tertiary and quaternary folding structures similar or essentially similar to the wild type protein (Thomas E. Creighton, Proteins, Second Edition, 1993, 232-236, ISBN 0-7167-2317-4). The differences of regenerated silk proteins in their protein folding pattern, especially for their tertiary and quaternary folding compared to native silk proteins have apparently no negative impact on their use as cosmetic or pharmaceutical ingredients (as described, for example, by Tsubouchi et al. in international patent application PCT/JP01/02250). However, for more demanding applications, such as the production of mechanically strong films, coatings and moulded objects or the biomimetic spinning of silks (as described by Vollrath and Knight in European Patent 1244828, assigned to Spintec Engineering GmbH), the correct folding and self-assembly of the silk proteins used for production of said materials plays a role in determining the mechanical strength and functional features of the formed materials. Due to the differences between the regenerated silk proteins and the native silk proteins noted above, the quality of regenerated silks has not been sufficient for the production of high quality silk materials through moulding, coating or biomimetic spinning as described in the above mentioned European patent 1244828.
European Patent Application No 1241178 (assigned to the National Institute of Agrobiological Sciences and Kowa Co) teaches a method for the production of silk fibroin by dissolving cocoons in an aqueous alkaline solution or an aqueous urea solution. The aqueous alkaline solution described in the examples of the '178 patent application is sodium carbonate or lithium thiocyanate at a pH of 7. Subsequently acetone or alcohol is added to the aqueous alkaline solution to precipitate the fibroin. The results shown in the patent application do not indicate that high molecular weight silk proteins exhibiting native tertiary and quaternary protein folding conformations were produced.
The '178 patent application reports (paragraph 18) also a method in which the silk gland is extracted from the body of a silkworm followed by extraction of the protein from a silk gland lumen. The disclosure suggests, however, that the process is not suitable for industrial production because the fibroin obtained contains impurities such as silkworm humor and silkworm gland cells.
A method for the extraction of native silk proteins from silkworm glands has been described in U.S. Pat. No. 7,041,797 (Vollrath, assigned to Spintec Engineering GmbH). The '797 patent describes an approach in which the silk glands are removed from the body of the silkworm followed by removal of an epithelial layer of the silk glands. The method works well for the manual extraction from individual ones of the silk glands. However, it is tedious and time consuming if a larger number of the silk glands need to be extracted. It is thus impracticable as a production method for the native proteins from the silk glands on an industrial scale. The inventor of the '797 patent has also not detailed a method or an apparatus which allows for the efficient and homogeneous mixing and pooling of proteins extracted from the silk glands as well as for the incorporation of additives in the extracted content from the silk gland.
Similarly Japanese Patent Application JP-A-2268693 (Asahi) teaches a method of cultivating a silk gland obtained from silkworms (such as from Bombyx mori) and using a culture medium. The culture medium is removed using dialysis to obtain an aqueous solution of the silk fibroin. However, the inventors in the '693 patent application did not consider how to incorporate additives homogenously into the highly viscous content of the silk gland.
A cruder method for obtaining fibroin protein is disclosed in Japanese Patent Application JP-A-3209399 (Terumo) in which the heads are cut off of grown silkworms. The fibroin protein is then harvested by pressing the abdomen of the silkworm to extract the fibroin protein. Sericin is removed from the resulting protein mixture by treating the protein mixture with a weak alkali, such as Na2CO3 The inventors of the '399 patent application did not teach how to include additives in the protein mixtures. Further the method of the '399 patent application has the disadvantage that incorporation of impurities from the silkworm body is not easily avoided. These impurities have to be removed or they may affect the properties of biomimetically spun fibers spun or of coated or moulded objects produced with said protein mixtures.